The present invention relates to a nucleic acid molecule encoding a (poly)peptide having chemotaxis inhibitory protein (CHIPS) activity. The invention further relates to the use of the information contained in the nucleic acid for the preparation of the corresponding (poly)peptide and to vectors and hosts for use therein. The invention in addition relates to non-(poly)peptide molecules having a similar structure and function as the (poly)peptides. The (poly)peptide having CHIPS activity that is encoded by the nucleic acid molecule of the invention can be used in the treatment of inflammation reactions. The (poly)peptides and non-(poly)peptides can in addition be used for inhibiting activation of leukocytes and endothelial cells.
Leukocytes are mainly involved in protecting the body against foreign invaders (e.g. bacteria, viruses, fungi, and cancer cells). The most important cells are lymphocytes, monocytes and neutrophils. Lymphocytes form the specific immune system and cause immune reactions against invaders. Their most important task is to build up specific memory against the invader, so that the next time the invader enters the body it is recognised, killed and removed rapidly. Sometimes these lymphocytes not only attack invaders, but also react against certain structures and/or molecules (so-called auto-antigens) of the own body, causing auto-immune diseases (e.g. rheumatoid arthritis). Killing and removal of invaders is mostly done by monocytes and neutrophils, cells of the innate immune system, by direct recognition of the invaders or with the help of specific lymphocytes.
In contrast to the delicate network of the fine-tuned and controlled reactions of lymphocytes, cells of the innate system react in a relatively non-specific and aggressive way. Since they are part of the body's first line of defence, their most important task is to kill and remove the invading agent as quickly as possible. This is accomplished through very aggressive substances (e.g. free radicals and enzymes) that are not only lethal to the invader, but also cause damage to host cells in the vicinity. Substances from these damaged cells and the locally activated cells from the innate system itself will further attract increasing numbers of neutrophils and monocytes, causing local inflammation. In most cases, such an aggressive and damaging inflammatory reaction, caused by overactivated neutrophils, is unnecessary. In some cases this inflammatory response is responsible for serious, sometimes lethal disorders and includes conditions like Adult Respiratory Distress Syndrome (ARDS), severe tissue damage following thrombotic events such as heart attacks and stroke, inflammatory bowel diseases and rheumatoid arthritis. The inflammation will subside once all the invaders have been killed and removed, together with the various cells killed in the process. Healing of the wound, caused by the inflammatory response, can then begin. Although there is some overlap in function, the main task of neutrophils is to attack the invaders and the main task of monocytes is to remove the debris resulting from this attack. In addition, neutrophils have another peaceful task in assisting the wound healing process.
When bacteria have invaded the body and, for example, infected the central nervous system (as in meningitis) they start to produce microbial substances, including the formylated polypeptides, such as, for example, the formyl-methionyl-leucyl-phenylalanine (fMLP) peptide. Other substances of microbial origin activate the complement factor 5 (C5) convertase enzyme-complex, that converts C5 of the complement system into its activated C5a form. Both C5a and fMLP are chemo-attractants: substances that can activate and attract cells from the blood vessels (the migration process). Neutrophils are responsive to these two substances and also to interleukin-8 (IL-8). This “chemokine” (the name given to chemo-attractants that are produced by cells of the immune system) is produced mainly by activated monocytes (but also in minute amounts by the activated neutrophils themselves). Neutrophils interact with these substances, because they have receptors for these substances on the outside of their cell membrane.
Activated neutrophils can easily migrate from blood vessels. This is because the chemo-attractants, microbial products and substances from activated monocytes will have increased the permeability of the vessels and stimulated the endothelial cells of the vessel walls to express certain adhesion molecules. Neutrophils express selectins and integrins (e.g. CD11b/CD18) that bind to these adhesion molecules. Once the neutrophil has adhered to the endothelial cells, it is able to migrate through the cells, under the guidance of chemo-attractants/chemokines, towards the site of infection, where the concentration of these substances is at its highest. These substances also activate neutrophils to produce a range of other molecules, some of which attract more neutrophils (and subsequently monocytes), but, mostly, they are responsible for destroying the invading bacteria. Some of these substances (e.g. free radicals, enzymes that break down proteins (proteases) and cell membranes (lipases)) are so reactive and non-specific that cells from the surrounding tissue (and the neutrophils themselves) are destroyed, causing tissue damage. This damage is exacerbated by the presence of blood derived fluid which has transgressed the leaky vessel wall and is responsible for the swelling that always accompanies inflammation (called oedema). The pressure build up caused by this excess fluid results in further cell damage and death.
Later in the inflammatory process, monocytes migrate to the scene and become activated. Besides their role in removing bacteria and cell debris, they also produce substances such as tumour necrosis factor (TNF) and IL-8, which in turn attract more activated neutrophils, causing further local damage. TNF also has a direct stimulatory effect on neutrophils. Once all the invaders have been removed, the inflammatory response will subside and the area will be cleared of the remaining ‘casualties’. Then the process of wound healing starts. Although it is known that neutrophils play a pivotal role in wound healing, it is not clear which neutrophil-derived substances are involved and how the neutrophils are active in healing without being aggressive to the surrounding tissue. In general, damaged tissue will be replaced by scar tissue formed mainly of fibroblasts and collagen. When inflammation occurs in areas of the body with an important function, like tissues formed from heart muscle cells, brain cells or lung alveolar cells, normal function will be compromised by the resulting scar formation, causing serious conditions like heart failure, paralysis and emphysema. To minimise the debilitating consequences of these conditions, it is important to ‘dampen’ the inflammatory reaction as quickly as possible.
Intervention to control the acute early phase inflammatory response presents an opportunity to improve the prognosis for a wide range of patients whose symptoms can be traced back to such an event. Such an approach has been advocated for many acute and chronic inflammation-based diseases and shown to have potential based on findings from relevant disease models. Classical anti-inflammatory drugs such as steroids and Non Steroid Anti-Inflammatory Drugs (NSAIDS) do not have the ideal profile of action, either in terms of efficacy or safety. Steroids affect the ‘wrong’ cell type (monocytes) and their dampening effects are easily bypassed. NSAIDS generally show a relatively mild effect partly because they intervene at a late stage in the inflammatory process. Both classes of drugs produce a range of undesirable side effects resulting from other aspects of their pharmacological activity. Drugs acting directly and specifically to prevent migration and activation of neutrophils may have a number of advantages. Several drugs under early development only interfere with one individual aspect of neutrophil activation (e.g. C5 convertase inhibitors, antibodies against C5a, C5a-receptor blocking drugs) and migration (antibodies against integrins (like. CD11b/CD18) and L-selectin on neutrophils and antibodies against adhesion molecules (like ICAM-1 and E-selectin) on endothelial cells). Antibodies against TNF and IL-8 have effects in chronic inflammation, but only marginal effects in acute inflammation, because of the minimal role monocytes (which are mainly responsible for these substances' production) play in the acute phase.
Sometimes, the cause of the acute inflammation cannot be removed and the inflammation becomes chronic. With the exception of tuberculosis, chronic hepatitis and certain other conditions, this is seldom the case with infections. However, chronic inflammation can also be caused by stimuli other than bacteria, such as auto-immune reactions. Research has shown that in chronic inflammation the role of monocytes is much more prominent, and that neutrophil migration and activation, monocyte migration and activation, tissue damage, removal of dead cells and wound healing are all going on at the same time. This complex cascade of interactions between cells and many different cytokines and chemokines has been the subject of intensive research for many years. It was believed that monocytes- and their products were the most important elements that needed to be inhibited to dampen chronic inflammation. This explains why steroids, which specifically interact with monocytes, are generally more effective in chronic as opposed to acute inflammation. Long-term treatment with steroids however, is not a desirable option, because severe and unacceptable side effects can occur at the doses required to produce a clinical effect. Newer treatments using antibodies against TNF or IL-8 have shown good results, and were initially seen as proof of the major role monocytes were thought to play in chronic inflammation. Recent research casts doubts on an exclusive role for monocytes in inflammation and points to a critical role for neutrophils, which are now seen to represent better targets for therapeutic intervention.
The underlying cause of a chronic inflammatory condition is not always clear, and the original cause may not always be responsible for future recurrence. Some scientists believe that in certain chronic inflammatory diseases there is a continuous cycle of events. Their idea is that existing activated neutrophils and monocytes continuously attract and activate new groups of cells, thus perpetuating the inflammatory response even when the initial stimulus is no longer present. This would suggest that an acute or periodic treatment with an effective inhibitor of the neutrophil and monocyte activation would stop the cycle of new cell recruitment, leading in due course to modification of disease progression, or even a complete cure, and not just symptomatic relief.
In the research that led to the present invention a new agent with inflammation-inhibiting properties was found in the extracellular medium of growing Staphylococcus aureus (S. aureus). This agent is the subject of co-pending application PCT/NL99/00442. The agent was found to be capable of directly or indirectly blocking different chemokine receptors. Incubation of different cells with the medium resulted in a greatly reduced expression of a number of the chemokine receptors, both of the expression of receptors of classical chemotactic agents such as fMLP and C5a on granulocytes and of the expression of CXCR4 and CCR5 receptors on lymphocytes, monocytes and macrophages. The reduced receptor expression was related to greatly reduced chemotaxis relative to the chemokines, as well as a reduced infection with HIV.
The activity of the protein is already manifest in the culture supernatant of the growing S. aureus. The active protein could be further purified, for example by means of a number of Ligand Dye columns. A pre-purification was first performed on a so-called “yellow column” (“Reactive Yellow 86” ligand dye cross-linked 4% beaded agarose column (Sigma)), followed by an absorption chromatography column (the so-called “green column” (“Reactive Green 19” ligand dye cross-linked 4% beaded agarose column (Sigma)) and a DNA column (DNA Cellulose (Pharmacia)). Both latter columns can be interchanged. The DNA column removes a contaminant with the same molecular weight as the protein. The absorption chromatography column concentrates the protein and is selective for the protein. Finally, a post-purification also takes place by means of gel filtration and anion exchange chromatography (MonoQ, Pharmacia). In the gel filtration the protein with the molecular weight of about 17 kDa is selected. This is the protein that was found to have chemotaxis inhibitory properties. Because this protein is isolated from the supernatant of the Staphylococcus aureus and gives inhibition of chemotaxis, this protein was named “CHIPS”: CHemotaxis Inhibitory Protein from Staphylococcus aureus (herein also referred to as the “original CHIPS”).
Isolation of the CHIPS protein out of the supernatant of S. aureus is not very cost-effective. In addition, it is desirable for the practical use of CHIPS in therapy that the active part of the protein is isolated. Smaller protein or peptide molecules have a reduced risk of inducing an immunological response in a subject receiving the protein or peptide for therapy. Furthermore, it is desirable to be able to modify the protein or peptide to further increase the biological activity and/or lower the immunogenicity thereof.
It is therefore the object of the present invention to provide the means for producing the original CHIPS protein or other corresponding (poly)peptides that have CHIPS activity, as well as functional fragments, derivatives or analogues thereof other than by isolation from the natural producing host cell.